Tissue engineering (TE) offers the potential to create replacement heart valves which have the potential for growth and remodeling, overcoming the limitations of current heart valve devices. Using autologous cells and biodegradable polymers, TE heart valves (TEHV) have been fabricated and have functioned in the pulmonary circulation of growing lambs for up to four months. Despite these promising results, significant questions remain. For example, the role of initial scaffold structure and mechanical properties to guide the development of optimal extra- cellular matrix (ECM) structure and strength are largely unexplored. While detailed biomechanical investigations of the in-vitro incubation process could shed much light on optimizing TEHV designs, little work has been conducted to date. Finally, our understanding of the structure-strength relations in native pulmonary valve (PV), which serves as the ultimate design paradigm, is profoundly incomplete. Our long-term goal is to develop a rigorous quantitative understanding of the biomechanical events that occur during in-vivo TEHV remodeling, and to use this knowledge to develop functionally equivalent TEHV designs. By functional equivalent we refer to the fact we aim to develop an engineered tissue that can perform an equivalent physiologic function (e.g. have requisite mechanical properties and durability) without having to precisely reproduce tri-layer cuspal structure. Prior to undertaking comprehensive in-vivo studies, we believe that detailed knowledge of the factors necessary for optimizing TEHV structure and biomechanics during in-vitro incubation must first be established. We hypothesize that precise control of 3D scaffold structure, initial scaffold mechanical properties and biodegradation rates, and well- controlled hemodynamic loading conditions can be used to optimize TEHV designs to duplicate native PV function. In addition, the structure-strength relations of the native pulmonic valve will be rigorously established in order to establish the TEHV design functional endpoint. We will explore our hypotheses with the following specific aims: 1) Quantify the shape of the ovine pulmonary outflow track and determine the mechanics of the native ovine PV cusp. 2) Quantify how initial scaffold structure, composition, degradation rates, and mechanical properties can be exploited to optimize the resultant engineered heart valve tissue. 3) Perform in-vitro evaluation of TEHV fabricated using optimal scaffold designs and 3D guided RV outflow track geometry using novel bioreactor loop imaging system.